Method for high efficiency protein delivery into plastids

ABSTRACT

The present invention provides a recombinant DNA molecule encoding a fusion protein, comprising a first DNA sequence encoding a high-efficiency transit peptide operably linked to a second DNA sequence encoding a passenger protein, wherein the high-efficiency transit peptide is selected from the group consisting of transit peptides of the precursors of translocon at the inner envelope membrane of chloroplasts 40 kD (prTic40), chaperonin 10-2 (prCpn10-2), Fibrillin 1B (prFibrillin), ATP sulfurylase 1 (prAPS1), ATP sulfurylase 3 (prAPS3), 5′-adenylylsulfate reductase 3 (prAPR3), stromal ascorbate peroxidase (prsAPX), prTic40-E2A (a prTic40 variant), prCpn10-1-ΔC7C37S (a chaperonin 10-1 variant), a functional fragment of any of the transit peptides and an equivalent thereof. And the present invention also provides a method of high efficiency delivery of a protein into plastids using the high-efficiency transit peptides.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Divisional of co-pending U.S. application Ser. No.15/210,445, filed on Jul. 14, 2016, which claims the priority benefit ofProvisional Application No. 62/192,393, filed on 14 Jul. 2015; theentire contents of all of the above-identified applications areincorporated herewith by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to plant biotechnology. More particularly,the present invention relates a method for high efficiency proteindelivery into plastids, in particular, leucoplasts.

2. The Prior Arts

Plastids are essential organelles in plants responsible for functionsranging from photosynthesis, biosynthesis of all fatty acids, starch,carotenoids, and most amino acids, to assimilation of nitrogen andsulfur. Plastids differentiate into different functional types indifferent tissues, for example chloroplasts in green tissues forphotosynthesis, chromoplasts in petals and fruits for carotenoidpigments accumulation, and leucoplasts in non-green tissues forsynthesis and storage of nutrients, including starch, proteins and oils.To perform these specific functions, different types of plastidsrequire, and therefore import, different proteins.

Although plastids have their own genome, most plastid proteins areencoded by the nuclear genome, synthesized in the cytosol as a largerprecursor with an N-terminal extension called the transit peptide.Transit peptides are necessary and sufficient for targeting passengerproteins into plastids, i.e., a transit peptide can be taken from theoriginal precursor and fused to a passenger protein and results indelivery of the passenger protein into plastids. Transit peptides thatcan deliver passenger proteins into chloroplasts with high efficiency,for example the transit peptide of RuBP carboxylase small subunitprecursor (prRBCS), have been identified and used to deliver passengerproteins into plastids.

Most grain- and root-type food crops, for example rice, corn andcassava, use leucoplasts to synthesize and store the starch that is usedto feed the majority of the world population. However, despite theeconomic importance of leucoplasts, almost all of our knowledge aboutplastid protein import is derived from studies with chloroplasts andlittle is known about how proteins are imported into leucoplasts.Unfortunately, leucoplasts clearly have a different substratepreference, and transit peptides like that of prRBCS import proteinspoorly into leucoplasts. For example, it has been shown that the transitpeptide of prRBCS could not direct the import of the passenger proteingreen fluorescent protein (GFP) into leucoplasts in endosperms oftransgenic wheat (Primavesi et al., 2008). Using leucoplasts isolatedfrom castor seeds and chloroplasts isolated from pea, it has been shownthat prRBCS imported much better into chloroplasts than into leucoplasts(Wan et al., 1996). Using leucoplasts and chloroplasts isolated from pearoots and leaves, respectively, it has been shown that prRBCS could notbe imported into leucoplasts at all (Yan et al., 2006). Nonetheless, inthese studies, no proper quantitative comparisons were performed so theimport efficiency of transit peptides could not be compared directlyamong one another. Therefore the exact import efficiency of prRBCStransit peptide into leucoplasts is not known and no transit peptideswith high leucoplast import efficiency have been discovered.

The transit peptide for prRBCS is the most widely used transit peptidefor delivering engineered proteins into plastids in biotechnologyapplications. Examples include the famous Golden Rice, Roundup Ready®corn and Dicamba resistant soybean. As discussed above, many reportshave suggested that this transit peptide deliver proteins poorly intoleucoplasts and therefore is not the best transit peptide forapplications that need to express proteins in leucoplasts. However, noreport has performed quantitative comparisons between transit peptides.If quantitative comparison can be performed and transit peptides withhigher leucoplast import efficiency than prRBCS transit peptide can beidentified, these new transit peptides would be valuable tools fordelivering engineered proteins into leucoplasts.

SUMMARY OF THE INVENTION

The present invention relates to a recombinant DNA molecule encoding afusion protein comprising a first DNA sequence encoding ahigh-efficiency transit peptide operably linked to a second DNA sequenceencoding a passenger protein, a DNA construct comprising saidrecombinant DNA molecule operably linked to a promoter, a plant materialcomprising said DNA construct or said recombinant DNA molecule, and amethod for high efficiency protein delivery into plastids, inparticular, leucoplasts, using said recombinant DNA molecule or saidconstruct.

Leucoplasts are plastids essential for the synthesis and storage ofstarch, lipids and proteins. Most grain and root-type crops useleucoplasts to synthesize and store nutrients. Modifications of thebiosynthesis processes within leucoplasts to better suit human needswill require high efficiency delivery of engineered proteins intoleucoplasts. Many reports have shown that proteins that could importefficiently into chloroplasts import poorly into leucoplasts because thetwo plastids have different protein preference. However, mostplastid-targeting transit peptides currently available are derived fromprecursor proteins with high chloroplast import efficiency. No transitpeptide with high leucoplast import efficiency is available. To identifytransit peptides with high leucoplast import efficiency, the presentinvention first optimized the in vitro leucoplast import system to allowfast, quantitative and accurate comparisons of import efficiencies of alarge number of plastid precursor proteins. Using this leucoplast importsystem, the present invention identified a group of nine precursorproteins that have very high import efficiency into leucoplasts andchloroplasts. These precursors imported into chloroplasts equally wellor better than prRBCS, but imported into leucoplasts two to eight timesbetter than prRBCS, whose transit peptide is currently the most widelyused transit peptide for directing engineered proteins into allplastids, including leucoplasts.

An objective of the present invention is to provide a recombinant DNAmolecule encoding a fusion protein, wherein said recombinant DNAmolecule comprising a first DNA sequence encoding a high-efficiencytransit peptide operably linked to a second DNA sequence encoding apassenger protein, wherein the high-efficiency transit peptide isselected from the group consisting of the transit peptides of theprecursors of translocon at the inner envelope membrane of chloroplasts40 kD (prTic40), chaperonin 10-2 (prCpn10-2), Fibrillin 1B(prFibrillin), ATP sulfurylase 1 (prAPS1), ATP sulfurylase 3 (prAPS3),5′-adenylylsulfate reductase 3 (prAPR3), stromal ascorbate peroxidase(prsAPX), prTic40-E2A (a prTic40 variant), prCpn10-1-ΔC7C37S (achaperonin 10-1 variant), a functional fragment of any of said transitpeptides and an equivalent thereof.

Another objective of the present invention is to provide a DNA constructcomprising the recombinant DNA molecule encoding a fusion protein asdescribed herein, operably linked to a promoter.

Another objective of the present invention is to provide a plantmaterial transformed with, and comprising the DNA construct.

Another objective of the present invention is to provide a method ofhigh efficiency delivery of a passenger protein into plastids of thetransformed plant material, comprising: providing a recombinant DNAmolecule encoding a fusion protein; linking the recombinant DNA moleculeoperably with a promoter to form a DNA construct; and transforming theDNA construct into a plant material to express the fusion protein,wherein the recombinant DNA molecule comprises a first DNA sequenceencoding a high-efficiency transit peptide operably linked to a secondDNA sequence encoding the passenger protein, wherein the high-efficiencytransit peptide is selected from the group consisting of the transitpeptides of the precursors of translocon at the inner envelope membraneof chloroplasts 40 kD (prTic40), chaperonin 10-2 (prCpn10-2), Fibrillin1B (prFibrillin), ATP sulfurylase 1 (prAPS1), ATP sulfurylase 3(prAPS3), 5′-adenylylsulfate reductase 3 (prAPR3), stromal ascorbateperoxidase (prsAPX), prTic40-E2A (a prTic40 variant), prCpn10-1-ΔC7C37S(a chaperonin 10-1 variant), a functional fragment of any of saidtransit peptides and an equivalent thereof.

In one embodiment of the present invention, the first DNA sequenceencoding a high-efficiency transit peptide is selected from the groupconsisting of SEQ ID NO:10 to SEQ ID NO:18, a functional fragment of anyof said SEQ ID NOs and an equivalent thereof.

In one embodiment of the present invention, the high-efficiency transitpeptides as described herein are capable of delivering an amount ofpassenger proteins that is equal or higher than the amount of passengerproteins a prRBCS transit peptide is capable of delivering intochloroplasts, and are capable of delivering an amount of passengerproteins that is higher than the amount of passenger proteins a prRBCStransit peptide is capable of delivering into leucoplasts.

In one embodiment of the present invention, the passenger protein is abiologically-active protein. In another embodiment, the passengerprotein is a protein to be delivered to plastids.

In one embodiment of the present invention, the promoter is functionalin a plant cell.

In one embodiment of the present invention, the plant material isselected from the group consisting of a plant cell, a plant tissue, aplant tissue culture, a callus culture and a transgenic plant.

In one embodiment of the present invention, the plant material isobtained from a monocotyledon or a dicotyledon plant.

Accordingly, the nine transit peptides of the present invention,including transit peptides of the precursors of the translocon at theinner envelope membrane of chloroplasts 40 kD (prTic40) (SEQ ID NO:10),chaperonin 10-2 (prCpn10-2) (SEQ ID NO:11), Fibrillin 1B (prFibrillin)(SEQ ID NO:12), ATP sulfurylase 1 (prAPS1) (SEQ ID NO:13), ATPsulfurylase 3 (prAPS3) (SEQ ID NO:14), 5′-adenylylsulfate reductase 3(prAPR3) (SEQ ID NO:15), stromal ascorbate peroxidase (prsAPX) (SEQ IDNO:16), prTic40-E2A (a prTic40 variant) (SEQ ID NO:17) andprCpn10-1-ΔC7C37S (a chaperonin 10-1 variant) (SEQ ID NO:18), provide avaluable tool to enable high efficiency delivery of engineered proteinsinto plastids for all kinds of plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show that prRBCS imported much less efficiently intoleucoplasts than into chloroplasts no matter whether the comparison wasdone based on equal proteins or on equal plastids, and prTic40 had muchhigher leucoplast import efficiencies than prRBCS. In FIG. 1A,leucoplasts and chloroplasts were isolated from roots of 4-day-old peaseedlings grown in the dark and leaves of 7-day-old pea seedlings grownunder 12-h photoperiod, respectively. The isolated leucoplasts (500 or113.67 μg protein) and chloroplasts (500 μg protein) were incubatedunder import conditions with 18 μL in vitro-translated [³⁵S]Met-prTic40or [³⁵S]Met-prRBCS and 3 mM ATP in import buffer in a final volume of200 μL for 25 min. The two different concentrations of leucoplasts, 500and 113.67 μg of protein, are for comparing with chloroplasts based onequal amounts of proteins and equal numbers of plastids, respectively.500 μg of leucoplasts have the same amount of proteins as 500 μg ofchloroplasts, while 113.67 μg of leucoplasts have the same number ofplastids as 500 μg of chloroplasts. After import, intact leucoplasts andchloroplasts were re-isolated through a 10% and 40% Percoll cushion,respectively. The import samples were analyzed by SDS-PAGE, stained withCoomassie blue, and dried for fluorography. 4% of the plastids in eachimport reaction (20 μg or 4.55 μg of proteins) were loaded. Tr, 1% ofthe in vitro-translated precursor proteins used in each import reaction.In FIG. 1B, the imported mature proteins in experiments as those shownin FIG. 1A were quantified and the import efficiencies were calculated.Data shown are mean±SD of three independent experiments. Cpt,chloroplasts; Leu, leucoplasts. pr, precursor protein; m, mature proteinderived after import.

FIGS. 2A and 2B show that different precursor proteins had differentplastid preference. The precursor prFd-protein A serves as an example ofhighly preferring chloroplasts. The precursor prPDH E1α serves as anexample of mildly preferring chloroplasts. The precursor prCpn10-2serves as an example of showing no preference and being able to importwell into both chloroplasts and leucoplasts. In FIG. 2A, isolatedleucoplasts (113.67 μg protein) and chloroplasts (500 μg protein) wereincubated with in vitro-translated prFd-protein A, prPDH E1α, orprCpn10-2 under import conditions for 25 min. After import, intactleucoplasts and chloroplasts were re-isolated through a 10% and 40%Percoll cushion, respectively. The import samples were analyzed bySDS-PAGE, stained with Coomassie blue, and dried for fluorography. 4% ofthe plastids in each import reaction were loaded. Tr, 1% (forprFd-protein A and prPDH E1α) or 1.2% (for prCpn10-2) of the invitro-translated precursor proteins used in the import reactions. InFIG. 2B, imported mature proteins in experiments as those shown in FIG.2A were quantified and the import efficiencies were calculated. Datashown are mean±SD of three independent experiments. Cpt, chloroplasts;Leu, leucoplasts. pr, precursor protein; m, mature protein derived afterimport.

FIGS. 3A to 3C show that nine precursor proteins had high importefficiencies into both leucoplasts and chloroplasts, and these nineprecursor proteins all had much higher leucoplast import efficiency thanprRBCS. In FIG. 3A, isolated leucoplasts (113.67 μg proteins) andchloroplasts (500 μg proteins) were incubated with in vitro-translated[³⁵S]Met-precursor proteins under import conditions for 25 min. Afterimport, intact leucoplasts and chloroplasts were re-isolated through a10% and 40% Percoll cushion, respectively. The samples were analyzed bySDS-PAGE, stained with Coomassie blue, and dried for fluorography. 4% ofthe plastids in each import reaction were loaded. Tr, 1.2% of the invitro-translated precursor proteins used in each import reaction. pr,precursor protein; m, mature protein derived after import. In FIGS. 3Band 3C, imported mature proteins in chloroplasts (3B) and leucoplasts(3C) in experiments as those shown in FIG. 3A were quantified and theimport efficiency of each precursor was calculated. The importefficiency of prRBCS was set as 1. Data shown are mean±SD of at leastthree independent experiments. Cpt, chloroplasts; Leu, leucoplasts.

FIGS. 4A and 4B show that the nine high-efficiency transit peptides ofthe present invention indeed delivered a higher amount of passengerproteins into leucoplasts than the prRBCS transit peptide. In FIG. 4A,protoplasts isolated from tobacco BY2 cells were co-transformed with GFPor transit-peptide-GFP fusion plasmids and the plasmid pBI221, whichdirected the expression of β-glucuronidase (GUS) in the cytosol andserved as an internal control for transformation and protein expressionefficiency. After transformation, the protoplasts were incubated in thedark at 25° C. for 16 h, analyzed by SDS-PAGE and immunodetected withantibodies indicated on the left. Thirty micrograms of proteins wereloaded in each lane. Plasmid DNA used for each lane is labeled at thetop. Lane 1, control protoplasts with no plasmid DNA added during thetransformation. Lane 2, protoplasts transformed with pBI221 and the GFPvector without fusing to any transit peptide. Lanes 3 to 12, protoplaststransformed with pBI221 and a plasmid encoding GFP fused to prRBCStransit peptide (lane 3), or GFP fused to one of the nine transitpeptides of the present invention (lanes 4 to 12). The transit peptidepart is indicated with subscript “tp”. Filled circles mark the precursorform of each fusion protein and brackets mark GFP imported into BY2 cellleucoplasts. In FIG. 4B, the amount of GUS and imported GFP inexperiments as those shown in FIG. 4A were quantified. The efficiency ofeach transit peptide was calculated by the amount of GFP normalized tothe amount of GUS in the same sample. The GFP/GUS ratio of RBCS_(tp)-GFPwas set as 1. Data shown are mean±SD of two independent experiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The accompanying drawings are included to provide a furtherunderstanding of the invention, and are incorporated in and constitute apart of this specification. The drawings illustrate embodiments of theinvention and, together with the description, serve to explain theprinciples of the invention.

As a first step in identifying precursors with high leucoplast importefficiency, the present invention optimized the in vitro leucoplastprotein import system for higher efficiency import and fast, accurateand quantitative comparisons, between leucoplasts and chloroplasts, andamong transit peptides. A group of nine precursors that imported intochloroplasts equally well as prRBCS, but imported into leucoplasts muchmore efficiently than prRBCS, were identified. The present inventionfurther showed that a higher amount of passenger proteins were importedinto leucoplasts in vivo when the passenger protein was fused to thesehigh efficiency transit peptides of the present invention, than whenfused to the prRBCS transit peptide.

Definition

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control.

As used herein, promoters suitable for the practice of this inventioninclude all promoters that have been shown to drive RNA expression inplants, including those that are constitutive, inducible ortissue-specific. Examples include, but are not limited to, maize RS81promoter, rice ubiquitin promoters, rice glutelin (Gt1) promoter, maizeRS324 promoter, maize PR-1 promoter, maize A3 promoter, maize L3 oleosinpromoter, rice actin promoters, prRBCS promoter, phytoene desaturasepromoter, sporamin promoter, gamma-coixin promoter, maize chloroplastaldolase promoter, nopaline synthase (NOS) promoter, octopine synthase(OCS) promoter, cauliflower mosaic virus (CaMV) 19S and 35S promoters,figwort mosaic virus 35S promoter, Arabidopsis sucrose synthasepromoter, R gene complex promoter, chlorophyll a/b binding protein genepromoter, CaMV35S promoter with enhancer sequences e35S, FMV35Spromoter, FLt36 promoter of peanut chlorotic streak virus, At.Act 7promoter, At.ANT1 promoter, FMV.35S-EF1a promoter, eIF4A10 promoter,AGRtu.nos promoter, and rice cytosolic triose phosphate isomerasepromoter.

As used herein, passenger proteins suitable for the practice of thisinvention are any protein the one of ordinary skill in the art wishes toexpress in plastids. Examples include, but are not limited to, GFP,β-glucuronidase (GUS), dicamba monooxygenase (DMO), enolpyruvylshikimate-3-phosphate (EPSP) synthase, glyphosate oxidase (GOX),phytoene synthase (psy), AtOR (R90H) (the R90H mutant of ArabidopsisORANGE protein), β-carotene ketolase and phytoene desaturase.

As used herein, “monocotyledon plant” refers to any of a class ofangiosperm plants having a single cotyledon in the seed, and includes(but is not limited to) rice, rye, wheat, barley, sorghum, maize, oat,orchids, lily, banana, taro and sugar cane.

As used herein, “dicotyledon plant” refers to an angiosperm that is nota monocotyledon, having two cotyledons in the seed, and includes (but isnot limited to) Arabidopsis, tobacco, potato, sweet potato, canola,soybean, bean, cotton, sunflower, white cauliflower, chrysanthemum,cassava and roses.

As used herein, a “high-efficiency” transit peptide refers to a transitpeptide which is capable of delivering an amount of passenger proteinsthat is equal or higher than the amount of passenger proteins a prRBCStransit peptide is capable of delivering into chloroplasts, and iscapable of delivering an amount of passenger proteins that is higherthan the amount of passenger proteins a prRBCS transit peptide iscapable of delivering into leucoplasts. The method used for comparingthe amounts of passenger proteins being delivered into different groupsof chloroplasts and leucoplasts can be the optimized method as describedherein, or any such method that is capable of differentiating between ahigher and a lower amount of a particular protein that has accumulatedin chloroplasts and leucoplasts of at least two experimental groups ofcells.

As used herein, the terms “construct” and “vector” are usedinterchangeably.

Transit peptides are known to function across species. For example, theprRBCS transit peptide from pea was used in the Golden rice creation.The pea prRBCS transit peptide is also used in creating the Dicambaresistant soybean and the initial studies for the creation wereperformed in Arabidopsis and tobacco. The prRBCS transit peptide fromArabidopsis was used in the Roundup Ready® corn (USDA, 1996). A transitpeptide from the waxy protein of corn was shown to function well inpotato. For the current invention, the high-efficiency transit peptideswere identified from pea and Arabidopsis and the initial tests wereperformed in pea, tobacco and rice and will also be tested inArabidopsis. It is expected that these nine transit peptides of thepresent invention will provide high efficiency delivery of proteins intoleucoplasts of all monocotyledon and dicotyledon plants, for exampleleucoplasts in the tubers, endosperms and roots of rice, barley, wheat,corn, cassava, potato and soybean and into leucoplasts of white coloredpetals of all flowers. Furthermore, because precursors containing thesetransit peptides also import very efficiently into chloroplasts, thesetransit peptides will also be expected to deliver proteins intochloroplasts with high efficiency.

EXAMPLE 1 Plastid Isolation, Protein Concentration Assays, and PlastidNumber Counting

Pea seedlings (Pisum sativum cv. Green Arrow) were grown at 20° C. onvermiculite. Leucoplasts were prepared from roots of 4- to 5-day-olddark grown seedlings as described (Chu and Li, 2015). Chloroplasts wereisolated from leaves of 7-day-old seedlings grown under a 12-hphotoperiod with a light intensity of approximately 150 μmol m⁻² s⁻¹ asdescribed (Perry et al., 1991), except 2 mM ascorbic acid, 0.1 mMdithiothreitol, and 1.2 mM glutathione were added in the grinding bufferused for homogenization. Isolated chloroplasts were adjusted to 1 mgchlorophyll mL⁻¹ in import buffer.

EXAMPLE 2 Optimization of the Protocol for Isolation of Import-CompetentLeucoplasts

To set up a quantitative leucoplast import system, the present inventionfirst optimized the conditions for leucoplast isolation. The presentinvention increased the concentration of EDTA and BSA in thehomogenization buffer and also added reducing agents into the buffer.After these modifications, the import efficiencies of precursor proteinsinto isolated leucoplasts were increased (Chu and Li, 2015).

EXAMPLE 3 Plasmid Construction and in vitro Translation of Precursorsfor in vitro Import into Isolated Plastids

Plasmids encoding prRBCS, prTic40, prFd-protein A, prPDH E1α, andprCpn10-2 have been described (Teng et al., 2012). The cDNA clones ofpda02149 for precursor of ATP sulfurylase 1 (prAPS1, AT3G22890) andpda04912 for precursor of 5′-adenylylsulfate reductase 3 (APR3,AT4G21990) were obtained from RIKEN BioResource Center. The leaf cDNApools of Arabidopsis thaliana (Columbia ecotype) were used as templatesto amplify the coding regions of Fibrillin 1B precursor (prFibrillin,AT4G22240), ATP sulfurylase 3 precursor (prAPS3, AT4G14680), and stromalascorbate peroxidase precursor (prsAPX, AT4G08390) with specific forwardand reverse primer pairs as described in Table 1. The PCR products ofprFibrillin were digested with HindIII and PstI and cloned into theHindIII/PstI of pSP72. The PCR products of prAPS3 and prsAPX weredigested with XhoI and SalI and cloned into the XhoI/SalI site of pSP72,respectively. The sequences of prFibrillin, prAPS3, and prsAPX wereconfirmed by sequencing and the plasmid was named pSP72-Fibrillin,pSP72-APS3, and pSP72-sAPX, respectively. Since the prFibrillin has onlytwo Met in the transit peptide region, the sequence encoding 2 extra Metwere inserted into the 3′end of cDNA before the stop codon using theQuikChange II Site-Directed Mutagenesis Kit (AGILENT TECHNOLOGIES) withprimers fibrillin-2M-F and fibrillin-2M-R (Table 1). The sequence wasconfirmed by sequencing and the plasmid was named pSP72-Fibrillin-2M andthis plasmid was used in the subsequent analyses. The prTic40 cordingregion in the pBS plasmid was digested with XhoI and PstI and clonedinto the XhoI/PstI site of pSP72 to generate the pSP72-Tic40 plasmid.This pSP72-Tic40 construct was used to generate the prTic40 variantusing the QuikChange II Site-Directed Mutagenesis Kit (AGILENTTECHNOLOGIES) with primers pSP72-Tic40-E2A-F and pSP72-Tic40-E2A-R(Table 1). The sequence was confirmed by sequencing and the plasmid wasnamed pSP72-Tic40-E2A. For the prCpn10-1 variant, prCpn10-1-ΔC7C37S, theprCpn10-1 plasmid (Teng et al., 2012) was used for site-directedmutagenesis via the QuikChange II Site-Directed Mutagenesis Kit (AGILENTTECHNOLOGIES) with primers Cpn10-1-ΔC7-F and Cpn10-1-ΔC7-R to generatethe plasmid pSP72-Cpn10-1-ΔC7 first. Then pSP72-Cpn10-1-ΔC7 was used forsite-directed mutagenesis with primers Cpn10-1-C37S-F andCpn10-1-C37S-R. The sequence was confirmed by sequencing and the plasmidwas named pSP72-Cpn10-1-ΔC7C37S. The in vitro expression of prAPS1 andprAPR3 was under the control of the T7 promoter. The in vitro expressionof prFibrillin, prAPS3, prsAPX, prTic40-E2A, and prCpn10-1-ΔC7C37S wasunder the control of the SP6 promoter.

[³⁵S]Met-labeled prPDH E1α was generated by in vitro transcription forsynthesizing RNA followed by in vitro translation using the RabbitReticulocyte Lysate system (PROMEGA). All other precursors weresynthesized using the TNT Coupled Wheat Germ Extract system or TNTCoupled Reticulocyte Lysate system (PROMEGA).

TABLE 1 Primers used for cloning in the invention PrimerNucleotide sequence Purpose fibrillin-F1- 5′-cgaagcttatggcgacggTo clone the coding region of HindIII tacaattgtc-3′prFibrillin into pSP72 SEQ ID NO: 21 fibrillin-R1-PstI5′-cgctgcagtcaaggattc To clone the coding region of aagagagg-3′prFibrillin into pSP72 SEQ ID NO: 22 APS3-F1-XhoI 5′-cactcgagatggcttccaTo clone the coding region of tgtccaccgtcttcc-3′ prAPS3 into pSP72SEQ ID NO: 23 APS3-R1-SalI 5′-cagtcgacttaaaccggaTo clone the coding region of atcttttccggaagtt-3′ prAPS3 into pSP72SEQ ID NO: 24 sAPX-F2-XhoI 5′-agctcgagatggcagagcTo clone the coding region of gtgtgtctc-3′ prsAPX into pSP72SEQ ID NO: 25 sAPX-R2-SalI 5′-gcgtcgacttagataacgTo clone the coding region of ataccctccg-3′ prsAPX into pSP72SEQ ID NO: 26 fibrillin-2M-F 5′-tctcttgaatcctatgatTo add two extra methionine gtgactgcaggtcg-3′residues in the C terminus of SEQ ID NO: 27 fibrillin fibrillin-2M-R5′-cgacctgcagtcacatca To add two extra methionine taggattcaagaga-3′residues in the C terminus of SEQ ID NO: 28 fibrillin pSP72-Tic40-E2A-F5′-cgataagcttgatatggc To mutate the glutamic acid gaatcttaacttagccc-3′residue at position 2 of SEQ ID NO: 29 prTic40 into alanine residuepSP72-Tic40-E2A-R 5′-gggctaagttaagattcg To mutate the glutamic acidccatatcaagcttatcg-3′ residue at position 2 of SEQ ID NO: 30prTic40 into alanine residue Cpn10-1-ΔC7-F 5′-gcttccactttcgtctctTo delete the cysteine residue ctaccaaatcct-3′at position 2 of prCpn10-1 SEQ ID NO: 31 Cpn10-1-ΔC7-R5′-aggatttggtagagagac To delete the cysteine residue gaaagtggaagc-3′at position 2 of prCpn10-1 SEQ ID NO: 32 Cpn10-1-C37S-F5′-cggaagtcgaagaggttc To mutate the cysteine residueccttagaatcaaagcga-3′ at position of 37 of prCpn10-1 SEQ ID NO: 33into serine Cpn10-1-C37S-R 5′-tcgctttgattctaagggTo mutate the cysteine residue aacctcttcgacttccg-3′at position of 37 of prCpn10-1 SEQ ID NO: 34 into serine

EXAMPLE 4 Protein Import into Isolated Plastids and Post-Import Analyses

After setting up the leucoplast isolation conditions, the presentinvention next compared the import behavior of various precursorproteins into leucoplasts and chloroplasts. An equal amount of proteins(Yan et al., 2006) or an equal number of plastids (Wan et al., 1996) wasusually used as the basis when comparing among import efficiencies ofdifferent plastids. To determine the number of plastids, the isolatedleucoplasts and chloroplasts were counted using the Multisizer 3 CoulterCounter (BECKMAN COULTER). The present invention used chloroplastsisolated from 7-day-old seedlings for robust import and for decreasingthe age difference between the leucoplast and chloroplast samples. Theaverage size of the isolated leucoplasts and chloroplasts was estimatedto be 1.81±0.21 μm and 3.24±0.23 μm, respectively. The same plastidpreparations were then used for protein concentration determination andthe average protein content was calculated to be 1.85±0.63 and 8.13±1.42μg/plastid for leucoplasts and chloroplasts, respectively. The presentinvention then used protein content as an estimate of plastid numbers inthe experiments thereafter. For example, 500 μg of plastid proteinswould represent approximately 2.71×10⁸ leucoplasts and 6.15×10⁷chloroplasts. For import comparison between chloroplasts and leucoplastson an equal protein basis, 18 μL [³⁵S]Met-labeled precursors wereincubated with isolated plastids equivalent to 500 μg plastid proteinsin the presence of 3 mM ATP in import buffer in a final volume of 200μL. For import comparison on an equal plastid number basis, 113.67 μgleucoplast proteins (2.71×10⁸ leucoplasts) and 500 μg chloroplastproteins (2.71×10⁸ chloroplasts) were used instead. Import reactionswere carried out at room temperature for 25 min and stopped bytransferring to a new tube containing 1 mL ice-cold import buffer. Theplastids were pelleted by centrifugation at 3,000 g at 4° C. for 3 minand re-suspended in 200 μL import buffer. The leucoplast suspensionswere underlaid with 1 mL 10% Percoll™ (v/v) in import buffer and thechloroplast suspensions were laid on top of a 40% Percoll™ (v/v) cushionto re-isolate the intact plastids with a swinging-bucket rotor by 2,900g for 6 min at 4° C. The plastids were washed once with import bufferand re-suspended in a small volume of import buffer. Proteinconcentrations of the plastid samples were measured with the BCA proteinassay kit (THERMO). Samples were analyzed by SDS-PAGE. Quantification ofgel bands was performed using the Fuji FLA5000 PhosphorImager (FUJIFILM,Tokyo).

EXAMPLE 5 prRBCS Imported Poorly into Leucoplasts and prTic40 ImportedEfficiently into both Chloroplasts and Leucoplasts

To provide quantitative comparison of the import efficiency of prRBCSinto chloroplasts and leucoplasts, import was compared based on equalproteins or on equal plastid numbers. The results showed that the importefficiency of prRBCS into leucoplasts was much lower than its importefficiency into chloroplasts no matter whether the comparison was doneon an equal amount of proteins or on an equal number of plastids basis(FIGS. 1A and 1B). Furthermore, in initial screenings from our originalcollection of precursor proteins, prTic40 showed the best importactivity into isolated leucoplasts and it was therefore used to comparewith prRBCS. prTic40 imported into chloroplasts with similar efficiencyto prRBCS, and imported into leucoplast much more efficiently thanprRBCS.

EXAMPLE 6 Identification of Transit Peptides with High Leucoplast ImportEfficiencies

The present invention further tested another three precursors,ferredoxin precursor (prFd), pyruvate dehydrogenase E1α subunitprecursor (prPDH E1α), and chaperonin 10-2 (prCpn10-2) precursor, toconfirm that different precursors have different plastid preferences.For prFd, the present invention used the construct prFd-protein A, whichcontains ferredoxin transit peptide fused to Staphylococcal protein A,as described (Smith et al., 2004). As shown in FIGS. 2A and 2B,prFd-protein A imported efficiently into chloroplasts but minimallyimported into leucoplasts, similar to the results of prRBCS. Incomparison, although prPDH E1α also imported more efficiently intochloroplasts, but its import into leucoplasts was about half that ofchloroplasts. In comparison, prCpn10-2 imported equally well into bothchloroplasts and leucoplasts.

Precursors like prTic40 and prCpn10-2 are of particular interestsbecause they can import very efficiently into both chloroplasts andleucoplasts. The present invention further cloned and tested the importof more than 60 plastid precursor proteins from Arabidopsis in theleucoplast import system the present invention set up. The presentinvention found another five precursors that also exhibited very highimport efficiency into both chloroplasts and leucoplasts (FIGS. 3A to 3Cand Table 2). They include precursors to Fibrillin 1B (prFibrillin), ATPsulfurylase 1 (prAPS1) and 3 (prAPS3), 5′-adenylylsulfate reductase 3(prAPR3) and stromal ascorbate peroxidase (prsAPX). Another twoprecursor variants, prTic40-E2A and prCpn10-1-ΔC7C37S, also showed highimport efficiency into both chloroplasts and leucoplasts (FIGS. 3B and3C and Table 2). For these nine precursors, their chloroplast importefficiency was comparable to that of prRBCS (FIG. 3B), but theirleucoplast import efficiency was 2˜8 times higher than that of prRBCS(FIG. 3C).

TABLE 2 Transit peptides with high import efficiencies in plastids.Amino acids of the transit peptide are shown in lowercaseletters and the first three amino acids in the mature regionare shown in capital letters. The prRBCS transit peptideused as a control is also shown here. Precursors (Accession Transitnumber) Full name Nucleotide sequences peptide sequence prTic40Translocon atggagaatc ttaacttagc ccttgtttct tcccctaaacmenlnlalvs spkplllghs (AY157668) at theccctgctttt aggacattcc tcctcaaaaa acgttttctc ssknvfsgrk sftfgtfrvsinner envelope aggaaggaag tctttcactt ttgggacgtt tcgcgtttctansssshvtr aaskshqnlk membrane ofgctaactctt catcctctca tgtcaccagg gctgcttcta svqgkvnahd FASchloroplasts 40 aatctcacca aaatctaaaa tctgtgcagg ggaaggtgaaSEQ ID NO: 10 kD tgcgcatgat tttgctagc SEQ ID NO: 1 prCpn10-2chaperonin 10-2 atggcttcga gtttcattac agtacctaaa cccttcttgtmassfitvpk pflsfpiktn (AT3G60210)ccttccccat caaaaccaat gctcctactc tacctcagca aptlpqqtll girrnsfringacccttctc ggaattcgaa gaaattcctt tagaattaac AVS gccgtttcc SEQ ID NO: 2SEQ ID NO: 11 prFibrillin 1B Fibrillin 1Batggcgacgg tacaattgtc cacccaattt agctgccaaa matvqlstqf scqtrvsisp(AT4G22240) ccagagtttc aatctcaccg aactctaaat ctatctccaansksiskppf lvpvtsiihr gcctccgttt ctggtaccgg tgacctcaat tattcaccgcpmistggiav sprrvfkvra ccgatgatct ccaccggagg aatcgctgtt tccccccgtatdtgeigsal laaEEA gagttttcaa agtccgagcc acagatacgg gagagataggSEQ ID NO: 12 atcagctcta ttggcggcgg aggaagca SEQ ID NO: 3 prAPS1 ATPatggcttcaa tggctgccgt cttaagcaaa actccattcc masmaavlsk tpflsqpltk(AT3G22890) sulfurylase 1 tctctcaacc actaaccaaa tcatctccaa actccgatctsspnsdlpfa aysfpskslr ccccttcgcc gcggtttcct tcccttccaa atccctacgcrrvgsiragl iapdggklve cgccgcgtag gatcaatccg agccggatta atcgctcccgliveepkrre kKHE acggtggtaa gcttgtagag ctcatcgtgg aagagccaaaSEQ ID NO: 13 gcggcgagag aagaaacacg ag SEQ ID NO: 4 prAPS3 ATPatggcttcca tgtccaccgt cttccccaaa ccaacctctt masmstvfpk ptsfisqplt(AT4G14680) sulfurylase 3 tcatctctca acctctaaca aaatctcaca aatccgattckshksdsvtt sisfpsnskt cgtaaccaca tccatttcat tcccttcgaa ttcgaaaactrslrtisvra gliepdggkl cgtagcttaa gaaccatctc tgtacgagct ggcttaatcgvdlvvpeprr rEKK agccagatgg tgggaaactt gtggatcttg ttgtaccggaSEQ ID NO: 14 accgagacgg cgagagaaga aa SEQ ID NO: 5 prAPR3 5′-atggcactag caatcaacgt ttcttcatct tcttcttctg malainvsss sssaissssf(AT4G21990) adenylylsulfate cgatctcaag ctctagcttc ccttcttcag atctcaaagtpssdlkvtki gslrllnrtn reductase 3aacaaaaatc ggatcattga ggttattgaa tcgtaccaat vsaaslslsg krssvkalnvgtctctgcgg cttctctgag tttgtccggg aagagatcct gsitkesiva sevtekldvvccgtgaaagc tcttaatgtg caatcaatta caaaggaatc EVEcattgttgct tctgaggtta cagagaagct agatgtggtg SEQ ID NO: 15 gaagttgaa SEQ ID NO: 6 prsAPX stromal atggcagagc gtgtgtctct cacactcaac ggaaccctccmaervsltln gtllsppptt (AT4G08390) ascorbatetttctcctcc tcccacaaca acaacaacaa caatgtcttc ttttmssslr sttaaslllrperoxidase ttctctccga tctaccaccg ccgcttctct tctcctccgc ssssssrSTLtcctcctcct cctcctccag atccactctc SEQ ID NO: 16 SEQ ID NO: 7 prTic40-E2ATranslocon atggcgaatc ttaacttagc ccttgtttct tcccctaaacmanlnlalvs spkplllghs (AY157668 at theccctgctttt aggacattcc tcctcaaaaa acgttttctc ssknvfsgrk sftfgtfrvsvariant) inner envelope aggaaggaag tctttcactt ttgggacgtt tcgcgtttctansssshvtr aaskshqnlk membrane ofgctaactctt catcctctca tgtcaccagg gctgcttcta svqgkvnahd FASchloroplasts 40 aatctcacca aaatctaaaa tctgtgcagg ggaaggtgaaSEQ ID NO: 17 kD variant tgcgcatgat tttgctagc SEQ ID NO: 8 prCpn10-chaperonin 10-1 atggcttcca ctttcgtctc tctaccaaat cctttctttgmastfvslpn pffafpvkat 1-ΔC7C37S variantcttttccggt caaagcaact actccttcga cggctaacca tpstanhtll gsrrgslrik(AT2G44650 tacgcttctc ggaagtcgaa gaggttccct tagaatcaaa AIS variant)gcgatttcc SEQ ID NO: 18 SEQ ID NO: 9 prRBCS Ribulose-1,5-atggcttcct caatgatctc ctccccagct gttaccaccg massmisspa vttvnragag(NM_001248385) bisphosphate tcaaccgtgc cggtgccggc atggttgctc cattcaccggmvapftglks magfptrktn carboxylasecctcaaatcc atggctggct tccccacgag gaagaccaac nditsiasng grvqcMQVsmall subunit aatgacatta cctccattgc tagcaacggt ggaagagtac SEQ ID NO: 20aatgcatgca ggtg SEQ ID NO: 10

EXAMPLE 7 Plasmid Construction for in vivo Expression of Fusion Proteinsin Tobacco BY-2 Suspension Culture Cells and Rice Calli

Plasmids for transient in vivo expression were prepared as follows. Thecoding region corresponding to the transit peptide and the first threeamino acids in the mature region of prRBCS, prTic40, prCpn10-2,prFibrillin, prAPS1, prAPS3, prAPR3, prsAPX, prTic40-E2A,prCpn10-1-ΔC7C37S (Table 2) were amplified by PCR using a forward primerthat added a XbaI site and a reverse primer that added a BamHI site tothe amplified fragment. The PCR fragment for each transit peptide wasdigested and cloned into the XbaI/BamHI site of the plasmid p326GFP (Leeet al., 2002), resulting in translational fusion of the transit peptideto the N terminus of GFP. The sequence was confirmed by sequencing andthe plasmid was named prRBCS_(tp)-GFP, prTic40_(tp)-GFP,prCpn10-2_(tp)-GFP, prFibrillin_(tp)-GFP, prAPS1_(tp)-GFP,prAPS3_(tp)-GFP, prAPR3_(tp)-GFP, prsAPX_(tp)-GFP, prTic40-E2A_(tp)-GFPand prCpn10-1-ΔC7C37S_(tp)-GFP, respectively. The transit peptide-GFPfusion constructs were placed under the control of the cauliflowermosaic virus 35S (CaMV35S) promoter and the nopaline synthase (nos)terminator. These transit peptide-GFP fusion constructs wereco-transformed with the plasmid pBI221, which contains CaMV35S drivenP-glucuronidase (GUS) gene and serves as an internal control fortransformation and protein expression efficiency. Protoplasts isolatedfrom tobacco BY-2 suspension cells were transformed by polyethyleneglycol mediated transformation. The amounts of GFP and GUS proteinproduced were determined by immunoblotting. The efficiency of eachtransit peptide was calculated by the amount of GFP produced normalizedto the amount of GUS produced for each transformation. Riceembryo-induced calli were transformed by particle bombardment andAgrobacteria-mediated transformation. Multiple transformationexperiments were performed for each construct and the average efficiencyof each transit peptide was calculated and compared to the averageefficiency of prRBCS transit peptide. The subcellular localization ofthe expressed proteins was confirmed by confocal microscopy.

EXAMPLE 8 Demonstration of Transit Peptide Efficiency by in vivoExpression of Fusion Proteins

The present invention further fused the transit peptides from the ninehigh-import-efficiency precursors to GFP as described above and testedthe in vivo leucoplast import efficiency of these nine fusion proteins.The present invention used transient expression in tobacco BY-2suspension culture cells and rice calli, which are widely used andrepresent established model systems for non-green leucoplast-containingtissues of dicotyledon and monocotyledon plants, respectively. Bothsystems can be easily transformed and served as a quick and reliabletool for evaluating the in vivo leucoplast import efficiency of thefusion proteins. The results (FIGS. 4A and B) showed that, when GFP wasfused to eight of nine transit peptides of the present invention, theamount of GFP imported into leucoplasts was much higher than when GFPwas fused to the transit peptide of prRBCS. One of the transit peptidesof the present invention had a similar efficiency to the prRBCS transitpeptide.

EXAMPLE 9 Plasmid Construction and Protein Expression in TransgenicArabidopsis Plants

The DNA fragment encoding GFP from the plasmid p326GFP (Lee et al.,2002) was amplified by PCR using a forward primer that added a BamHIsite and a reverse primer that added an XbaI site to the amplifiedfragments. The fragment was digested and cloned into the BamHI/XbaI siteof pSP72. The sequences were confirmed by sequencing and the plasmid wasnamed pSP72-GFP. The DNA fragments encoding the transit peptide and thefirst three amino acids of the mature region of prRBCS, prTic40,prCpn10-2, prFibrillin, prAPS3, prAPR3, prsAPX, prTic40-E2A,prCpn10-1-ΔC7C37S (Table 2) were amplified by PCR using a forward primerthat added a Sad site and a reverse primer that added a BamHI site tothe amplified fragment. The fragments were digested and cloned into theSacI/BamHI site of pSP72-GFP to generate the transit peptide-GFP fusionconstructs. The DNA fragment encoding the transit peptide and the firstthree amino acids of the mature region of prAPS1 was amplified by PCRusing a forward primer that added a Kpnl site and a reverse primer thatadded a BamHI site to the amplified fragment. The fragments weredigested and cloned into the KpnI/BamHI site of pSP72-GFP. All sequenceswere confirmed by sequencing. The DNA fragments encoding the transitpeptide-GFP were excised by SacI/PstI (for prRBCS_(tp)-GFP,prTic40_(tp)-GFP, prCpn10-2_(tp)-GFP, prFibrillin_(tp)-GFP,prAPS3_(tp)-GFP, prAPR3_(tp)-GFP, prsAPX_(tp)-GFP, prTic40-E2A_(tp)-GFPand prCpn10-1-ΔC7C37S_(tp)-GFP) and KpnI/PstI (for prAPS1_(tp)-GFP) andcloned into the SacI/PstI site and KpnI/PstI site of the binary vectorpCHF1 (Hajdukiewicz et al., 1994), respectively. The transit peptide-GFPfusion constructs were placed under the control of the CaMV35S promoterand the RBCS terminator. The resulting plasmids were transformed intoAgrobacterium tumefaciens GV3101. Arabidopsis (Columbia ecotype) plantswill be transformed by the floral spray method (Chung et al., 2000).Transgenic plants harboring the introduced transit peptide-GFP fusiontransgene will be identified on MS medium containing 100 μg/mL G418.Multiple independent transgenic plants will be obtained for each transitpeptide-GFP construct. The RNA and protein amount of the expressed GFPwill be detected by quantitative RT-PCR and immunoblotting. Thelocalization of GFP in root leucoplasts will be verified by confocalmicroscopy. The size of GFP should also be the processed mature-size GFPon immunoblots, corroborating with delivery to plastids and removal ofthe transit peptide. After normalization to the GFP RNA level, theefficiency of each transit peptide in delivering GFP to root leucoplastsin each transgenic plant will be calculated. The average efficiency ofeach transit peptide will be compared to the average efficiency ofprRBCS transit peptide. The efficiency of each transit peptide indelivering GFP to leaf chloroplasts will also be compared to that ofprRBCS transit peptide. It is expected that the efficiency of each ofthe high-efficiency transit peptides as described herein, in deliveringGFP to chloroplasts, will be equal or higher than that of prRBCS transitpeptide. It is further expected that the efficiency of each of thehigh-efficiency transit peptides as described herein, in delivering GFPto leucoplasts, will be higher than that of prRBCS transit peptide.

The present invention provides a group of nine transit peptides thathave very high import efficiency into both chloroplasts and leucoplasts.They are transit peptides of the precursors of translocon at the innerenvelope membrane of chloroplasts 40 kD (prTic40), chaperonin 10-2(prCpn10-2), Fibrillin 1B (prFibrillin), ATP sulfurylase 1 (prAPS1), ATPsulfurylase 3 (prAPS3), 5′-adenylylsulfate reductase 3 (prAPR3), stromalascorbate peroxidase (prsAPX), prTic40-E2A (a prTic40 variant) andprCpn10-1-ΔC7C37S (a chaperonin 10-1 variant). These nine transitpeptides of the present invention all have similarly highchloroplast-import efficiency, and much higher leucoplast-importefficiency, than the prRBCS transit peptide. The more than 60 precursorsthe present invention tested and the nine precursors of the presentinvention have never been tested for leucoplast import before. Thetransit peptide of prRBCS is the most widely used transit peptide fordelivering engineered proteins into plastids. Examples include thefamous Golden Rice, Roundup Ready® corn and Dicamba resistant soybean.In the case of Golden rice, for example, they have used the prRBCStransit peptide to deliver the bacterial carotene desaturase into ricegrain leucoplasts. The present invention has shown quantitatively herethat, for delivering proteins into leucoplasts, the nine transitpeptides of the present invention are much more efficient than theprRBCS transit peptide. If the one of ordinary skill in the art had usedone of the transit peptides from the present invention, the productionof provitamin A in the rice grains may be even higher. The next step isto test which of the nine transit peptides offer the highest proteinimport efficiency into plastids in stably transformed plants.

Although the present invention has been described with reference to thepreferred embodiments thereof, it is apparent to those skilled in theart that a variety of modifications and changes may be made withoutdeparting from the scope of the present invention which is intended tobe defined by the appended claims.

What is claimed is:
 1. A recombinant DNA molecule encoding a fusionprotein, wherein said recombinant DNA molecule comprises a first DNAsequence encoding a transit peptide operably linked to a second DNAsequence encoding a passenger protein, wherein the transit peptideconsists of the first 40 amino acids of SEQ ID NO:11.
 2. A DNA constructcomprising the recombinant DNA molecule according to claim 1, whereinsaid recombinant DNA molecule is operably linked to a promoter.
 3. TheDNA construct according to claim 2, wherein the promoter is functionalin a plant cell.
 4. A plant material comprising the DNA constructaccording to claim
 3. 5. The plant material according to claim 4,wherein the plant material is selected from the group consisting of aplant cell, a plant tissue, a plant tissue culture, a callus culture anda transgenic plant.
 6. The plant material according to claim 4, whereinthe plant material is obtained from a monocotyledon or a dicotyledonplant.
 7. A method of delivering a passenger protein into leucoplasts,comprising: (a) providing a recombinant DNA molecule encoding a fusionprotein; (b) linking the recombinant DNA molecule operably with apromoter to form a DNA construct, wherein the promoter is functional ina plant cell; and (c) introducing the DNA construct into a plantmaterial to express the fusion protein; wherein the recombinant DNAmolecule comprises a first DNA sequence encoding a transit peptideoperably linked to a second DNA sequence encoding a passenger protein,wherein the transit peptide consists of the first 40 amino acids of SEQID NO:11.
 8. The method according to claim 7, wherein the plant materialis selected from the group consisting of a plant cell, a plant tissue, aplant tissue culture, a callus culture and a transgenic plant.
 9. Themethod according to claim 7, wherein the plant material is obtained froma monocotyledon or a dicotyledon plant.